Vectashields containing dapi

Leg discs from prepupae (from 0h to 4h after puparium formation depending on experiments) are dissected in PBS 1x. Tissue are fixed by paraformaldehyde 4% diluted in PBS 1x during 20 minutes. Then the samples are washed and saturated in PBS 1x, 0.3% triton x-100 and BSA 1% (BBT). Next, the samples are incubated overnight at 4°C with primary antibodies diluted in BBT. Samples are washed for 1h in BBT before a 2h incubation at room temperature with secondary antibodies diluted in BBT. Finally, samples are washed with PBS 1x, 0.3% Triton x-100 for 1h and mounted in Vectashields containing DAPI (Vector Laboratories). A 120-μm-deep spacer (Secure-Seal™ from Sigma-Aldrich) is placed in between the glass slide and the coverslip to preserve morphology of the tissues.
Primary antibodies from Developmental Studies Hybridoma Bank (DSHB) are klarsicht-C antibody (9C10-s, mouse, 1:50), Lamin Dm0 (ADL195-s, mouse, 1:50) and E-Cadherin antibody (DCAD2, rat, 1:50). Anti-cleaved Dcp-1 (#9578, rabbit, 1:200) was obtained from Cell Signaling Technologies. Secondary antibodies (Alexa488 and 647) are purchased from Interchim and diluted at 1:200 or 1:100 respectively. Phalloidine-Rhodamine (Fischer Scientific) used to stain F-actin is diluted at 1:200.

Primary antibodies obtained from Developmental Studies Hybridoma Bank were: rat anti-E-Cad (DCAD2, 1/50) and rat anti-α-Catenin (DCAT-1, 1/50). Rabbit anti-Snail antibody was a gift from Leptin. Secondary antibodies coupled to Alexa-488, -555 and -647 were obtained from Fisher Scientific and diluted 1/200. Samples were mounted in Vectashield with DAPI (Vector Laboratories).
Leg discs from prepupae were dissected in PBS 1×. Tissues are fixed by paraformaldehyde 4% diluted in PBS 1× during 20 min.
Embryos were fixed for 5 min in heptane:formaldehyde 37% (1:1), then devitellinized manually and stained immediately.
After fixation, the samples were washed and saturated in PBS 1×, 0.3% triton x-100 and BSA 1% (BBT). Next, the samples were incubated overnight at 4 °C with primary antibodies diluted in BBT. Samples were washed for 1 h in BBT before a 2 h incubation at room temperature with secondary antibodies diluted in BBT. Finally, samples were washed with PBS 1×, 0.3% Triton x-100 for 1 h and mounted in Vectashields containing DAPI (Vector Laboratories). A 120-µm-deep spacer (Secure-SealTM from Sigma-Aldrich) was placed in between the glass slide and the coverslip to preserve morphology of the tissues.